Review and discuss the significance of animal cloning (such as: Dolly the sheep)

Animal Cloning is the production of one or more identical copies of a genome of a particular animal. Embryo splitting (or blastomere separation) and nuclear transfer are two methods by which animals may be cloned. Both of these methods require gestation in the uterus to attain complete development. Embryo splitting is the process whereby an embryo has its cells (blastomeres) separated soon after fertilization at the 2nd, 4th or 8th cell stage. At this stage, each cell is considered to be at a stage where the genetic material has not been programmed to develop into a select tissue, it is undifferentiated and can therefore develop into a variety of tissues, or a complete organism given the correct conditions. This is referred to as totipotency and it allows scientists to split animal embryos into several cells to produce multiple organisms that are genetically identical.

Modern techniques begin with stripping the embryo of the protective layer. After each blastomere has been separated from the embryo mass, the cell is encased in its own protective synthetic layer. Each blastomere cell, is now considered a new separate embryo and is cultured in vitro and later in vivo in a surrogate mother until birth (Roberge, 2004).

Cloning by nuclear transfer is based on the concept that the animal’s genome is located in the cell nucleus. The only exception to this is the small amount of DNA of 16, 000 base pairs found in the mitochondria. In this process the genetic material from one cell is place into a recipient unfertilized egg which had its genetic material removed by enucleation. It is then necessary to activate the cell to start dividing. In mammals the egg must be artificially placed in the womb of a surrogate mother where it will grow until birth. During the mid 1980s, the first mammals were cloned by this process, thirty years after the initial experiments with frogs (Van Eenennam, n.d). Many reports of successful nuclear transfer experiments in mammals have been made, nearly all of them using cells taken directly from early embryos.

Two types of nuclear transfer that are used to clone animals are the Roslin Technique and the Honolulu technique. The Roslin technique is a variation of the somatic nuclear transfer, and this method was used to create Dolly. In this process, somatic cells with their nuclei are allowed to grow and divide, these cells are then deprived of nutrients to induce the cells into a suspended or dormant stage. An egg cell that has had its nucleus removed is then placed in close proximity to a somatic cell and both cells are shocked with an electrical pulse. The cells fuse and the egg is allowed to develop into an embryo.

The Honolulu Technique was developed at the University of Hawaii by Dr. Teruhiko. In this method, the donor nucleus from a somatic cell is removed using a special pipette, and is microinjected into an egg that has had the nucleus removed. The egg is placed in a chemical solution and it is cultured. The developing embryo is then implanted into a surrogate and allowed to develop. Twenty-two fertile, female mice were cloned from nuclei of adult ovarian cumulus cells by this method.

Dolly the sheep was the first mammal to be cloned from an adult cell. She was derived from cells that were taken from the udder of a six year old ewe. In this case, the genetic material in the nucleus was transferred from adult somatic cells that were cultured. Individual cells were then fused with unfertilised eggs from which the genetic material had been removed. Two hundred and seventy seven of these 'reconstructed eggs' were cultured for 6 days in temporary recipients. Twenty-nine of the eggs that appeared to have developed normally to the blastocyst stage were implanted into 13 surrogate Scottish Blackface ewes. One gave rise to a live lamb, Dolly, 148 days later.

The Nuclear transfer technique was first reported in frogs in 1952 and has been used widely since in amphibians to study early development. In 1977, the cloning of mice using nuclei from early embryos was reported, however this work was not repeatable. Research on nuclear transfer however continued in cattle, this was driven by the prospect of large commercial benefits from multiplying elite embryos.

This is a preview of the whole essay

In the early 1980’s the former Animal Breeding Research Organisation started research aimed at producing transgenic sheep and cattle that would secrete human proteins in their milk. By the middle of the 1980’s the number of sheep have been cloned by several research groups around the world. This was done by transferring nuclei directly from early embryos. This process allows cloning to be carried out on an adult animal with known characteristics. A wide range of adult animal tissues have been used to successfully clone many species and this has a great significance.

The primary significance of animal cloning is probably in the opportunities it opens for genetic manipulation. Animal cloning allows for the generation of groups of genetically identical animals for research purposes, the rapid propagation of desirable animal stocks, to improvement in the efficiency of generating and propagating transgenic livestock, the production of targeted genetic alterations in domestic animals and the research in cell differentiation.

Dolly and other animals cloned from adult cells allow researchers to address developments that were not possible before. For example, most mammalian genetic research is currently being conducted in mice because their embryos are the easiest to manipulate. However, the findings in mice are not always applicable to humans because of their biological differences. Mammals such as sheep are biologically similar to humans, therefore the prospect of engineering sheep may lead the way to better animal models of human diseases, and other progress in research.

In recent years the agricultural industry has been trying to improve nuclear transfer cloning to facilitate the breeding of desirable livestock and biotechnology companies are investigating ways to explore the use of nuclear transfer cloning to improve the production of therapeutic drugs. In addition to drug production, an understanding of the details of nuclear translation cloning might lead to new therapies to treat human disease. There will be quality upgrades in terms of what animal produce naturally such as cows milk, and what they can artificially produce to cure serious disease.

Since animal cloning can be carried out on an adult cell with known characteristics, the best traits can be perpetuated and animals. These animals can also be used in large scale production of medically important proteins. Polly is a transgenic lamb and she can produce milk containing factor IX protein, which is the protein that is deficient in haemophiliacs. Dr John Clark and colleagues were able to direct expression of human genes specifically to the mammary gland, using the beta-lactoglobulin promoter. This success led to the setting up in 1987 of PPL Therapeutics and their subsequent production of Tracy, a transgenic sheep that secreted 35g of a human protein (alpha-1-antitrypsin) in each litre of her milk (Anon, n.d).

Another aspect of cloning that would be beneficial is the interest in producing pigs whose genes have been altered to produce a component of the human immune system's regulatory proteins. The process of using animal organs for human organ transplantation is called xenotransplantation. This would make pig organs more suitable for human transplantation. Such treatments are in progress, for example, heart valves of pigs in treating humans with malfunctioning hearts.

Cloning by nuclear transfer could also help generate gene alteration in animals much more efficiently. This would be of great significance for research in genetics and gene therapy. Introducing specific mutations in mice, called "knockout mice," has been an important way to study the effects of certain genes. Producing such animals requires that embryonic stem cells are altered in culture to produce a blastocyst, which is then placed in a surrogate. The resulting animal must then mate again to produce germline transmission of the altered stem cells. Cloning would allow the genes of large mammals to be altered in this way while accelerating the traditionally long generation and litter cycles.

Another means by which animal cloning can aid in research is in the efficient production of genetically identical animals. The production of these genetically identical, homozygous animals will aid in the elimination of genetic variation as a confounding variable in experiments. With nuclear transfer cloning, a single blastocyst can be used as the source of donor nuclei for subsequent blastocyst, which in turn can serve as donor nuclei. While the number of possible cycles is limited, nuclear transfer cloning would be an efficient way of carrying out the process. Generating such homozygous inbred lines in larger animals is difficult and time consuming because of the long gestation times and small numbers of offspring. The concept of generating small groups of identical animals by nuclear transfer has been proposed as an alternative strategy to obtaining a genetically identical group of animals.

In animal breeding, the rapid spread of traits within stocks of domestic animals is of commercial importance. Embryo transfer and artificial insemination can increase the reproductive output of individual selected male and female animals and are widely used in the livestock industry. The process of nuclear transfer cloning, especially from somatic cell nuclei, could provide an additional means of expanding the number of chosen livestock. One of the features unique to nuclear transfer technologies is the ability to make identical copies of adult elite cows, sheep, and pigs. This process may well be used in livestock production, if the efficiencies of adult nuclear transfer can be improved. However, it should be noted that the net effect of multiplying chosen animals by cloning will be to reduce the overall genetic diversity in a given livestock line, likely with negative long-term consequences. If this technique were to become widespread, efforts would have to be made to ensure a pool of genetically diverse animals for future livestock maintenance.

Cloning has further advanced the field of biotechnology in discovering new alternatives and methods to treating new diseases. Cloning will enable animal models that test theories of disease and treatment to be more easily developed before applying such applications in humans.

One of the benefits of animal cloning is in the area of pharmaceutical production. The ability to clone will allow for animals to be genetically engineered for a particular protein following mass production. The milk of livestock animals can be modified to contain large amounts of proteins which are important for pharmaceutical production such as insulin or factor VIII for treatment of human disease by expressing human genes in the mammary gland (Houdebine, 1994 cited by Anon n.d). In sheep greater than 50 percent of the proteins in milk can be the product of a human gene (Colman, 1996 cited by Anon n.d). The milk of even transgenic mice can yield large (milligrams) quantities of recombinant proteins. Since many such proteins are active at very low concentrations, it is estimated that production of human drugs from transgenic animals could be considerably more cost-effective than current methods being used. (Anon, n.d).

The production of the first transgenic lamb produced by nuclear transfer was Polly, in July 1997. In this case, the donor cell was a foetal fibroblast that had been transfected with the gene coding for human blood clotting factor IX ( cited by Anon n.d).

The expression of human antigens in pigs could alleviate organ shortages by minimizing or eliminating the rejection of pig organs transplanted into humans. However, other barriers such as the possibility of transmitting viruses from pigs to humans must be overcome. The current method of directly injecting genes into fertilized eggs is not very efficient the method used is by pronuclear injection. This procedure involves the introduction of 200-300 copies of the transgene into a recently fertilized egg which is then implanted in a surrogate mother. Not all injected eggs will develop into transgenic animals, and then not all transgenic animals will express the added gene in the desired manner. Only 2-3% of eggs injected give rise to transgenic offspring and only some of these express the added gene at sufficiently high levels to be of commercial interest. It is also only possible to add genes by pro-nuclear injection. It would be possible to carry out specific genetic modifications such as the removal or substitution of specific genes if animals can be derived from cells in culture. This has been achieved in mice using embryonic stem (ES) cells. In 1995 when Keith Campbell, Ian Wilmut and colleagues produced live lambs Megan and Morag by nuclear transfer from cells from early embryos that had been cultured for several months in the laboratory ( cited by Anon n.d).

The production of transgenic livestock is slow and expensive. Nuclear transfer would speed up the expansion of a successful transgenic line, and probably more importantly, it would allow more efficient generation of transgenic animals. Foreign DNA, such as a human gene, could be introduced into cell lines in culture and cells expressing the transgene could be characterized and used as a source of donor nuclei for cloning, and all offspring would likely express the human gene. If a human gene such as that for insulin could be expressed in the mammary gland, the milk of the sheep would be an excellent source of insulin to treat diabetes. In addition therapies for treating haemophilia have been investigated.

One of the reasons for carrying out cloning experiments is because there is need for a nontoxic agent to regulate gene expression in malignant cells. These experiments are designed to reveal the nature of the genetic material of the cancer cell and the capacity of that malignant genetic material to give rise to normal cell progeny. Cloning may be the most direct method of acquiring information about these two important aspects of cancer-cell biology (Nguyen, Peter).

Cloning provides a means whereby scientists will be given the tools to fully understand the biology of cancerous cells allowing them to find a safer alternative to current methods of treating cancer. In using cloning research on lab animals, scientists are beginning to find less traumatic treatments for cancer. Concurrently, researchers are already “modifying the genetic structure of animals in order to study illnesses and to generate human proteins and antibodies”(Wilson 463 cited by Nguyen, Peter n.d).

Cloning has benefits ecologically in that populations of rare endangered species may be saved. Nuclear transfer and techniques such an “embryo cloning” could contribute to the preservation of endangered species such as the Sumatran Tiger. Around the world scientists have begun collecting and storing frozen tissue samples of endangered animals for which cloning could prove to be a last resort. Work has already progresses towards cloning the Sumatran Tiger and China’s Giant Panda using the same technology that created Dolly the sheep.

Although cloning has many benefits, there are also many biological disadvantages. One such biological problem is the high mortality rate, however it is said that with greater understanding of what determinants cause the death of clones, the mortality may decrease. Another biological problem is the decline in species diversity. Ethical issues also arise which need to be addressed. There are numerous applications that nuclear transfer cloning might have for biotechnology, livestock production, and new medical approaches. Work with genetic manipulation and embryonic stem cells provide many pharmaceutical proteins and prospects for regeneration and repair of human tissues.

Referneces

Nguyen, P. Cloning, ethics, what lies ahead. (Maech 19, 2005)

Van Eenennam, A. (n.d) Livestock Cloning. (March 21, 2005)

Roberge, L. (October 18, 2004) Cloning: Scientific, Technological and Ethical Considerations. (March 21, 2005)

Anon. (n.d) The science and application of cloning. (March 20, 2005)

History-

Anderson B. et al. (2002) A Guide to Modern Science:Science and Technology in Today’s World, pp 112-115, Cloning, Fog City Press, San Francisco,CA